Estrogen hormone action within target tissues involves the interaction of the hormonal substance with two different receptor proteins termed ER-alpha and ER-beta. The specificity and responsiveness of tissues to hormonal stimulation are governed in most part by the presence and biochemical action of these receptor proteins. Estrogen receptors (ER) are thought to play a crucial role in development, reproduction and normal physiology. We have generated using gene targeting lines of mice homozygous for the disrupted ERa gene (aERKO) and ERb genes (bERKO)and the recent generation of double ERKO mice abERKO) and characterization of phenotypes in the reproductive, cardiovascular, immune, skeletal and neuroendocrine systems are currently underway. RPA analysis detected comparable levels of ER-b mRNA in tissues of aERKO mice suggesting that ER-b expression is not dependent on ERa. aERKO mice were totally unresponsive to uterotropic assays with estradiol, hydroxy TAM, DES. Further support came from the failure of estrogen, EGF or IGF-1 treatment to induce DNA synthesis in aERKO uteri, even though EGF and IGF-1 signaling was shown to be intact by stimulation of c-fos, or IRS-1 protein phosphorylation. Progesterone receptor mRNA was detected in aERKO mice, but not stimulated by estrogen in the uterus, mammary gland and ovary, indicating an estrogen dependent and independent gene regulation. aERKO females are infertile and have hypoplastic uteri and hyperemic ovaries with no apparent corpora lutea. The aERKO ovarian phenotype occurs developmentally and can be reversed by a GnRH antagonist. Ovarian gonadotropin receptor levels, serum estrogen, androgen and LH levels are elevated compared to WT and bERKO females. aERKO but not bERKO females have a loss in estrogen protection from ischemia and atherosclerosis. Findings in bERKO females assessed by continuos breeding studies and superovulation show arrested folliculogenesis and subfertility. Because of this phenotype we are evaluating whether mutations or polymorphism in the ER-beta gene may correlate with clincal cases of female infertility. Analysis of the mammary glands of adult aERKO females showed a primitive ductal rudiment rather than the fully developed ductal tree seen in WT or bERKO mice. aERKO were used as an experimental model system for evaluating the role of ERa in normal physiological endocrine function and hormonal carcinogenesis. Since aERKO mice have no mammary gland development, a test for the role of estrogen receptor in development of mammary tumors was developed. A cross of aERKO with a mammary sensitive mouse line (WNT-1) expressing the WNT-1 oncogene was produced. These data indicate that the WNT-1 oncogene does not require functional estrogen receptor to produce an epithelial cell specific mitogenic response. Additionally, these animals do develop tumors at approximately half the level as WT. These findings would oppose one earlier hypothesis that mammary tumors develop from hormone responsive tissues. aERKO males are also infertile, with a progressive lesion resulting in atrophy of the testes and seminiferous tubule dysmorphogenesis producing decreased spermatogenesis and inactive sperm. Sperm transplantation studies of aERKO males rescues the infertility phenotype indicating the need for ER-alpha was not in the germ cell but in the somatic cells of the male reproductive tract. The findings also clearly demonstrate a requirement for estrogen receptor action in normal male reproductive physiology. Males have reduced bone density and some alterations in cardiovascular function. Phenotypic differences were seen in sex and aggressive behavior in both aERKO males and females compared to the patterns in WT or bERKO mice. In contrast to the aERKO, the bERKO males are fertile with normal sexual behavior. Recent development of a viable abERKO shows a unique ovarian phenotype of transdifferentiation of granulosa to sertoli cells, suggesting that both ER signal mechanisms must be functional in order to maintain the proper differentiation state of the granulosa cells. Further characterization of the mice and comparison of the individual and double ER gene KO phenotypes will be required to more fully understand the physiological consequences of ER mediated actions and the specific roles of the two different forms of ER in estrogen hormone responsiveness. As a means of extending the ERKO model evaluating the mechanism of environmental estrogen action ERKO mice were treated with genistein which also acts as a tyrosine kinase inhibitor and shows that uterine growth was not stimulated indicating genistein acts through the estrogen receptor for this response. However, suppression of lutenizing hormone (LH) was shown to be as effective in ERKO as WT mice indicating this estrogen response by genistein does not require the receptor as previously thought. Differences in gene responses to hormonal xenobiotics were also seen in some in vitro gene regulation studies. Using transactivation assay system trichlorobiphenol was shown to be more active than estradiol on the lactoferrin gene. Use of this estrogen responsive yeast system also tested the xenobiotic estrogen synergy concept and showed that using a variety of genes that synergy was not apparent and had no universal application to hormone responsive systems.